PROJECT SUMMARY/ABSTRACT The goal of Project 2 is to understand how factors uniquely recruited to the mutant TERT promoter (TERTp) together with factors that are native to the wildtype (WT) promoter activate TERT to achieve tumor cell immortality. TERTp mutation occurs in nearly all glioblastoma (GBM) and oligodendroglioma (OD), enabling tumor cells to become immortal. We showed that the mutations allow the GA-binding protein (GABP) to aberrantly activate the mutant TERTp across many cancer types. In our preliminary data and new publication, reduction of GABP in GBM causes decreased TERT and a gradual and nearly complete loss of viability in a TERTp mutation-dependent manner. GABP is not normally present at the TERTp, however little else is known about this newly discovered central node in tumor cell immortality. We have discovered two novel candidate molecules - RNF2, an ubiquitin ligase that appears to drive activation of the mutant TERTp; and the tumor suppressor CIC that represses WT and mutant TERTp, but is recurrently mutated in OD and downregulated in GBM. Here, we will test the hypothesis that activation of the mutant TERTp and tumor immortality by GABP involve critical contributions from mutant allele-specific factors and native factors. In Aim 1, we will define the role of mutant-specific recruitment of RNF2 in promoting TERT expression and immortality. We will examine RNF2 recruitment to, and regulation of mutant TERTp across GBM and OD cultures, determine if knockdown of RNF2 alters telomerase activity, telomere length and tumor cell viability in vitro and tumorigenesis in vivo, and if combined inhibition of GABP and RNF2 accelerates these processes. Potential resistance mechanisms in tumors that grow despite reduced GABP will be addressed. In Aim 2, we will determine how WT and mutant CIC regulates TERT expression and immortality. WT CIC suppresses transcription several ETS factors which we previously demonstrated normally activate the WT and mutant TERT promoter. We will test CIC suppression of TERT expression across GBM and OD cultures, then test whether the recurrent loss of function mutations found in OD upregulate TERT, and which ETS factors mediate the effect. We will knockdown the CIC-regulated ETS factors and determine if they alter telomerase activity, telomere length and tumor cell immortality. In Aim 3, we propose to more broadly discover novel regulators of immortality using an in vivo CRISPRi screen. The gene inhibition will be modeled in TERTp mutant GBM cells with and without GABP- editing in order to identify factors that regulate cellular immortality in vivo either independently of or synergistically with GABP. Our results in glioma will build upon the central node of TERT-GABP interaction which we discovered, and may be relevant to the wider spectrum of TERTp mutant tumor types. By contrasting mechanisms of TERT regulation in two clinically distinct glioma subtypes -OD and GBM- we will identify TERTp regulatory mechanisms that are shared, or subtype-specific and potentially linked to the different patient outcomes. The tumor cell specific regulators may present new therapeutic opportunities.